Serum proteome profiles in cats with chronic enteropathies

Abstract Background Serum protein biomarkers are used to diagnose, monitor treatment response, and to differentiate various forms of chronic enteropathies (CE) in humans. The utility of liquid biopsy proteomic approaches has not been examined in cats. Hypothesis/Objectives To explore the serum proteome in cats to identify markers differentiating healthy cats from cats with CE. Animals Ten cats with CE with signs of gastrointestinal disease of at least 3 weeks duration, and biopsy‐confirmed diagnoses, with or without treatment and 19 healthy cats were included. Methods Cross‐sectional, multicenter, exploratory study with cases recruited from 3 veterinary hospitals between May 2019 and November 2020. Serum samples were analyzed and evaluated using mass spectrometry‐based proteomic techniques. Results Twenty‐six proteins were significantly (P < .02, ≥5‐fold change in abundance) differentially expressed between cats with CE and controls. Thrombospondin‐1 (THBS1) was identified with >50‐fold increase in abundance in cats with CE (P < 0.001) compared to healthy cats. Conclusions and Clinical Importance Damage to the gut lining released marker proteins of chronic inflammation that were detectable in serum samples of cats. This early‐stage exploratory study strongly supports THBS1 as a candidate biomarker for chronic inflammatory enteropathy in cats.

obtained via gastrointestinal endoscopy or surgery for histopathologic assessment are the current standard required for definitive diagnosis. 1,4 When diagnoses are not reached with histopathological evaluation, additional diagnostic tests such as immunohistochemistry and clonality testing might be required in ambiguous cases. 1 However, even with immunohistochemistry and clonality testing, definitive diagnoses are not always achieved. 1,5 There is a high rate of false positive samples with a specificity of 33% with clonality testing in cat samples. 5,6 Biomarkers have been investigated as potential diagnostic tests for CE with minimally invasive diagnostic methods. 7 The most commonly used biomarkers in cats in clinical practice are serum cobalamin and folate which can be misleading for diagnosis of CE in cats if comorbidities are present. 1 In humans, serum protein biomarkers are used to diagnose, monitor treatment response, and to differentiate between different forms of CE and therefore represent a novel, noninvasive, diagnostic, and monitoring tool for human IBD. [8][9][10][11][12][13] In cats, proteomics has been studied in several areas including chronic kidney disease, mammary carcinoma, pancreatic disease, osteoarthritis, and cat semen. [14][15][16][17][18][19] Given the extensive use of serum protein profiles in IBD in human medicine and studies in other areas of small animal medicine, serum proteomics might provide useful biomarkers in cats with CE. 8,14 The discovery of new proteomic biomarkers is a multiphase process which involves screening for potential biomarkers using an untargeted approach with a small sample size at the discovery phase followed by verification of selected proteins using targeted approaches with a larger sample size at the validation phase. 20,21 A recent study investigated the intestinal mucosal proteome in cats with 9 proteins found to be differentially expressed between healthy cats and cats with IBD and LGAL. 22 However, western blot analysis did not confirm significant differential protein expression. 22 The utility of serum proteomics in diagnosis of CE in cats has not been explored.
The aim of this exploratory study was to investigate changes in the serum proteomic profiles of cats with CE. This study provides useful preliminary data on the serum protein markers differentiating healthy cats from cats with CE. The ability to use a minimally invasive diagnostic method to assist in diagnosis of CE might reduce the need for more invasive diagnostics and anesthesia for pets with comorbidities and lead to reduced costs for pet owners.

| Animals and sample collection
Cases were recruited from 3 veterinary hospitals, including referral and primary care practices, between May 2019 and November 2020. Cats presented for evaluation of CE with clinical signs (vomiting, diarrhea, weight loss, or a combination of these signs) of at least 3 weeks' duration, and that were eventually diagnosed with CE/LGAL, were prospectively recruited for this study. Cat information including signalment, diagnostic tests, diet, previous and current treatment, and comorbidities was recorded. Cats with comorbidities such as pancreatitis, cholangiohepatopathy, urinary tract disease, and endocrinopathies, and cats without intestinal biopsies were excluded from further assessment. Diagnoses of CE were made on the basis of clinical signs, histopathology and exclusion of other causes of gastrointestinal manifestations, including metabolic disease, infection, parasitic disease, hepatic, and renal disease. The following diagnostic tests were performed: a complete blood count, a serum biochemistry profile, serum total T4 concentration, serum concentrations of cobalamin, abdominal ultrasonography and fecal flotation and PCR (feline coronavirus, Tritrichomonas foetus, Cryptosporidium species, panleukopenia virus, Clostriudium perfringens, Giardia species, Salmonella species, Toxoplasma gondii, Campylobacter jejuni, Campylobacter coli).
Serum feline pancreatic lipase immunoreactivity (fPLI) and feline trypsin like immunoreactivity (fTLI) were measured in some cats. All the cats diagnosed with CE using endoscopic biopsies were scored using the feline chronic enteropathy activity index (FCEAI) which was calculated based on their clinical signs, clinicopathological, and endoscopic findings. 3 Cats diagnosed via surgical biopsy were not FCEAI scored. All endoscopies were performed by a single board-certified veterinary internist (LB). Eight out of 10 abdominal ultrasounds were performed by board-certified veterinary radiologists from a single referral hospital while 2 ultrasound examinations were performed by general practitioners (cases recruited from primary care practices). All histopathological examinations of biopsied tissue samples were performed by boardcertified anatomic pathologists from different institutions and 7 cases were retrospectively reviewed following the WSAVA standards (MK).
Controls were healthy cats that did not show clinical signs of gastrointestinal disease or weight loss confirmed with history collection and unremarkable physical examination findings. Some control cats were staff cats while others were enrolled during annual wellness health check at primary care practices. Blood samples were collected via jugular venipuncture, followed by centrifugation for serum separation. The serum was then collected into a serum tube and stored at À20 C before analysis.

| Protein sample preparation
All serum samples were assessed for total protein concentration using the 2D Quant kit (Cytiva, Massachusetts, USA) as per manufacturer's instruction. Samples of 100 μg total protein were mixed in 50 μL AMBIC buffer (50 mM Ammoniumbicarbonate, 10 mM DTT, 2 M urea at pH 8) and trypsin digested at 25 C for 16 hours in a 1:100 enzyme-to-protein ratio based on the calculated serum protein concentration. 11 Digestion was halted by acidification. Each sample was then dried to remove the AMBIC, reconstituted in 50 μL 0.1% formic acid and desalted and nonpeptide contaminants removed using C18 stage tips (Thermo Scientific, Illinois, USA) according to the manufacturer's recommendations except that the elution buffer consisted of 80% CH 3 CN, 0.1% Formic acid.

| Mass spectrometry
Digested peptides were reconstituted in 10 μL 0.1% formic acid and separated by nano-LC using an Ultimate 3000 HPLC and autosampler (Dionex, Amsterdam, Netherlands) and followed methods described previously. 23

| Protein characterization
Protein dataset-peak lists were generated from raw files using Mascot Identifications were accepted if they could establish less than 5% false discovery rate (FDR) and contained at least 2 identified peptides per protein. 24

| Statistical analysis
Descriptive statistics of cat information including age, sex, weight, FCEAI and cobalamin concentrations were analyzed using R software.
Continuous data with non-normal distribution including age, weight and protein concentrations was compared and analyzed using the Mann-Whitney U test. Sex status was analyzed using Chi-square test.
Data were Log 2 transformed using NCSS (version 9) software and normal distribution confirmed by D'Agostino's K-squared test.
Changes in expression of protein (defined as fold change) were analyzed using Fisher's exact test with Benjamini-Hochberg adjusted P values. Differences in fold change were considered statistically significant at P < .02, with a minimum fold change of ≥5. [25][26][27] Fold change ratios and significance were calculated for CE/Control. For bioinformatics analysis, the Gene Ontology concept was used to investigate molecular function, biological processes, and cellular components. The identified biological module groups were evaluated by their enrichment score and the significance of the module's enrichment was determined by AmiGo Panther analysis. 28 Data were also analyzed with STRING version 11.5 database (https://string-db.org). 29 Proteins identified with significant (and fold change >2) changes in expression were uploaded into STRING to map corresponding protein-protein interactions. Graphical networks for these proteins were constructed based on their connectivity algorithms. mesenteric lymph node liver, or a combination of these tissues. The median number of biopsy specimens examined per site was 4 (range, [1][2][3][4][5][6][7][8][9][10][11]. Six cases were classified as lymphoplasmacytic enteritis (4 mod  between the CE and clinically healthy cats. There was also no T A B L E 2 Consolidated and significant differential proteins between cats with CE and controls (CE/C) were found in 26 proteins with fold change ≥5 (P < .02). Using log2 transformed fold change, the significantly changing proteins between control and cats with CE based on total spectral counts, 2 peptide identification and FDR of 5%, P ≤ .05 is shown in

| DISCUSSION
Proteomic analysis allows large scale detection and rapid identification of proteins of pathological significance. 30   nisolone and with Cushing's syndrome. 41,42 In our study, no correlation was found between THBS1 abundance and cats treated with prednisolone in all groups.
A further finding in the study reported here was the involvement of coagulation factors in enteropathies. Coagulation factor V showed a significant difference with >100-fold increase in relative abundance (P < .001) in cats with CE compared to controls in our analysis. Coagulation factor V is a protein of the coagulation system and acts as a non-enzymatic cofactor for activated factor X to form the prothrombinase complex which contributes to generation of thrombin. 43 The relationship between hemostasis and CE is complex. Increased risk of thromboembolism and platelet activation have been observed in IBD in humans. 44 Further, increased generation of thrombin has been demonstrated in human IBD patients compared to controls and was thought to be associated with a partial loss of function of the natural anticoagulant pathways. 44,45 The imbalance between procoagulant and anticoagulant pathways with the changes in the fibrinolytic system are thought to contribute to thromboembolism in human IBD. 44 A hypercoagulable state has also been detected by thromboelastography in dogs with normoalbuminemic and hypoalbuminemic chronic inflammatory enteropathy. 46 The relationship between proteins in our study was mapped using the STRING algorithm (Figure 3), and the findings were dominated by the involvement of ECM proteins. ECM remodeling is a hallmark of IBD in humans. 51 Recent studies support the role of the ECM as an active component in promoting inflammation in the pathogenesis of IBD. 51 The alteration of ECM in IBD is characterized by inflammatory mediators and activated matrix metalloproteinase-9 (MMP9), which increases intestinal permeability, epithelial apoptosis and loss of goblet cells in colitis in humans. 51 In dogs, upregulation of mucosal active MMP2 and MMP9 was found in the intestines of dogs with CE compared to healthy dogs. 52 In addition, intestinal stricture formation and fibrosis have been linked to increased production in specific components of the ECM including fibronectin and collagens in IBD. 51 In our study, many ECM components such as fibronectin, inter-alpha-trypsin inhibitor heavy chains, profilin-1 and actin were upregulated in cats with CE compared to healthy cats.
Further, as shown in the data reported here (Figure 3), the interrelationship between inflammation, ECM and platelets contributes to the upregulation of protein clusters involving ECM and altered coagulation. The dysregulation of vascular ECM in human IBD patients has been found to promote adhesion and extravasation of leukocytes and platelets in the endothelium. 51 The cell adhesion molecules such as E-selectin interact with components of the ECM (fibronectin, collagens, laminin) to regulate the recruitment of circulating leukocytes and control endothelial permeability. 51 An apparent change in lipid metabolism, with upregulation of apolipoproteins A-I, B-100, C-I, C-III, and E, was identified in cats with CE compared to control cats in our study. Of these proteins apolipoproteins B-100, C-I, C-III, and E showed fold change ≥5. In cats, the distribution of serum lipoproteins and apolipoproteins is unlike that observed in humans. Lipoproteins in the cats are larger and richer in triglycerides compared to humans. 53  In the study reported here, cats with CE had increased levels of other known acute phase reactants such as complement proteins C1 and C3. The complement system comprises of many plasma proteins that function as receptors or regulators of complement activation through 3 activation pathways: the classical pathway, lectin pathway, and the alternative pathway. 58 Complement activation leads to the formation of C3 convertase which is the major and most abundant component in the complement cascade. 58 Hyperactivation of complement has been reported in chronic inflammatory diseases and immunological diseases in people. 58,59 Furthermore, in people with Crohn's disease, alterations in complement cascade and innate immune response have been described. 60 Serum C3 and C4 complement components were increased in human patients with Crohn's disease, with higher levels found in human patients with active disease compared to inactive disease. 61 The increased fold change in complement proteins in cats with CE in our study could be related to the immunological basis of IBD or inflammation. Nevertheless, further studies are needed to establish the link between the complement system and CE in cats.
There are several limitations in our study. First, the small sample size was a limiting factor. Although differences in protein profile were observed between cats with CE and LGAL, we did not perform further analysis comparing the 2 groups due to the small sample size of cats with LGAL and therefore a definitive conclusion of difference in protein profiles between CE and LGAL cannot be drawn. Second, as the management of each case was at the discretion of the F I G U R E 4 A dot plot showing the relative abundance based on spectral count of THBS1 in individual control and cats with chronic enteropathies (CE). The median is illustrated by the black line, median for cats with CE is 4.5 (range, 0-21) and median for controls is 0 (range, 0-1).
attending veterinarian, not all diagnostic tests were performed in each cat and treatment was not standardized. Specifically, fecal PCR, flotation, fPLI and fTLI results were only available for some cats.
Although our analysis did not show any correlation in THBS1 abundance in cats treated with prednisolone, we cannot completely